基于Te-OnAdvanced的肺癌细胞15-脂氧化酶-2基因表达载体的构建与表达

目的：构建肺癌细胞15-脂氧化酶-2（15-Lox-2）的可诱导性真核表达载体pTRE-Tight-15-Lox-2,并在肺癌细胞中检测其是否可受强力霉素（DOX）诱导表达。方法：pcDNA3-15-LOX-2载体经＆0RI和XbaI双酶切线性化,回收15-LOX-2cDNA片段,将其克隆入pTRE-Tight载体的EcoRI和XbaI位点;采用脂质体法将pTet-0n-Ad-vanced与构建的pTRE-Tight-15-Lox-2共转染肺腺癌A549细胞,DOX诱导表达后,Western印迹检测15-Lox-2的表达水平。结果：构建了pTRE-Tight-15-Lox-2诱导表达载体;Western印迹检测表明,该载体能在肺癌细胞内表达,且其表达受DOX调控。结论：Tet-OnAdvanced系统能严密高效地调控15-LOX-2在肺癌细胞中的表达,为进一步研究15-LOX-2在肺癌中的作用奠定了基础。     

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of MDR1 ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDRl ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzedin vitro andin vivo; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDRl ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effective treatment method for patients with CEA-producing lung cancers which was usually refractory to conventional chemotherapy     

TFPI1 Mediates Resistance to Doxorubicin in Breast Cancer Cells by Inducing a Hypoxic-Like Response

Thrombin and hypoxia are important players in breast cancer progression. Breast cancers often develop drug resistance, but mechanisms linking thrombin and hypoxia to drug resistance remain unresolved. Our studies using Doxorubicin (DOX) resistant MCF7 breast cancer cells reveals a mechanism linking DOX exposure with hypoxic induction of DOX resistance. Global expression changes between parental and DOX resistant MCF7 cells were examined. Westerns, Northerns and immunocytochemistry were used to validate drug resistance and differentially expressed genes. A cluster of genes involved in the anticoagulation pathway, with Tissue Factor Pathway Inhibitor 1 (TFPI1) the top hit, was identified. Plasmids overexpressing TFPI1 were utilized, and 1% O₂ was used to test the effects of hypoxia on drug resistance. Lastly, microarray datasets from patients with drug resistant breast tumors were interrogated for TFPI1 expression levels. TFPI1 protein levels were found elevated in 3 additional DOX resistant cells lines, from humans and rats, indicating evolutionarily conservation of the effect. Elevated TFPI1 in DOX resistant cells was active, as thrombin protein levels were coincidentally low. We observed elevated HIF1α protein in DOX resistant cells, and in cells with forced expression of TFPI1, suggesting TFPI1 induces HIF1α. TFPI1 also induced c-MYC, c-SRC, and HDAC2 protein, as well as DOX resistance in parental cells. Growth of cells in 1% O₂ induced elevated HIF1α, BCRP and MDR-1 protein, and these cells were resistant to DOX. Our in vitro results were consistent with in vivo patient datasets, as tumors harboring increased BCRP and MDR-1 expression also had increased TFPI1 expression. Our observations are clinically relevant indicating that DOX treatment induces an anticoagulation cascade, leading to inhibition of thrombin and the expression of HIF1α. This in turn activates a pathway leading to drug resistance.     

Altered Expression of Proliferation-Inducing and Proliferation-Inhibiting Genes Might Contribute to Acquired Doxorubicin Resistance in Breast Cancer Cells

This study was designed to investigate the molecular changes that may develop during exposure of breast cancer cells to anticancer agents and that may lead to acquired resistance. We used two breast cancer cell lines, a parental (MCF7/WT) and a doxorubicin-resistant (MCF7/DOX) one. Cell survival, cell cycle distribution and RT-PCR expression level of genes involved in DNA damage response, MDR1, GST and TOPOIIα were measured. MCF7/DOX cells were five-fold more resistant to doxorubicin (DOX) than the MCF7/WT cells. DOX treatment causes arrest of MCF7/DOX cells in G1 and G2 phases of cell cycle whereas MCF7/WT cells were arrested in S-phase. The molecular changes in both cell lines due to DOX treatment could be classified into: (1) the basal level of p53, p21, BRCA1, GST and TOPOIIα mRNA was higher in MCF7/DOX than MCF7/WT. During DOX treatment, the expression level of these genes decreased in both cell lines but the rate of down-regulation was faster in MCF7/WT than MCF7/DOX cells. (2) The expression level of MDR1 was the same in both cell lines but 48 and 72 h of drug treatment, MDR1 disappeared in MCF7/WT but still expressed in MCF7/DOX. (3) There was no change in the expression level of BAX, FAS and BRCA2 in both cell lines. Conclusively, after validation in clinical samples, overexpression of genes like BRCA1, p53, p21, GST, MDR1 and TOPOIIα could be used as a prognostic biomarker for detection of acquired resistance in breast cancer and as therapeutic targets for the improvement of breast cancer treatment strategies.     

Chloride channel-3 mediates multidrug resistance of cancer by upregulating P-glycoprotein expression

Chloride channel-3 (ClC-3), a member of the ClC family of voltage-gated Cl⁻ channels, is involved in the resistance of tumor cells to chemotherapeutic drugs. Here, we report a new mechanism for ClC-3 in mediating multidrug resistance (MDR). ClC-3 was highly expressed in the P-glycoprotein (P-gp)-dependent human lung adenocarcinoma cell line (A549)/paclitaxel (PTX) and the human breast carcinoma cell line (MCF-7)/doxorubicin (DOX) resistant cells. Changes in the ClC-3 expression resulted in the development of drug resistance in formerly drug-sensitive A549 or MCF-7 cells, and drug sensitivity in formerly drug-resistant A549/Taxol and MCF-7/DOX cells. Double transgenic MMTV-PyMT/CLCN3 mice with spontaneous mammary cancer and ClC-3 overexpression demonstrated drug resistance to PTX and DOX. ClC-3 expression upregulated the expression of MDR1 messenger RNA and P-gp by activating the nuclear factor-κB (NF-κB)-signaling pathway. These data suggest that ClC-3 expression in cancer cells induces MDR by upregulating NF-κB-signaling-dependent P-gp expression involving another new mechanism for ClC-3 in the development of drug resistance of cancers.     

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of MDR1 ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDR1 ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzed in vitro and in vivo ; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDR1 ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effe     

Sensitization of multidrug-resistant malignant cells by liposomes co-encapsulating doxorubicin and chloroquine through autophagic inhibition

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters play a key role in the development of multidrug resistance (MDR) in cancer cells. P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) are important proteins in this superfamily which are widely expressed on the membranes of multidrug resistance (MDR) cancer cells. Besides, upregulation of cellular autophagic responses is considered a contributing factor for MDR in cancer cells. We designed a liposome system co-encapsulating a chemotherapeutic drug (doxorubicin hydrochloride, DOX) and a typical autophagy inhibitior (chloroquine phosphate, CQ) at a weight ratio of 1:2 and investigated its drug resistance reversal mechanism. MTT assay showed that the IC₅₀ of DOX/CQ co-encapsulated liposome in DOX-resistant human breast cancer cells (MCF7/ADR) was 4.7?±?0.2?μM, 5.7-fold less than that of free DOX (26.9?±?1.9 μM), whereas it was 19.5-fold in doxorubicin-resistant human acute myelocytic leukemia cancer cells (HL60/ADR) (DOX/CQ co-encapsulated liposome 1.2?±?0.1?μM, free DOX 23.4?±?2.8?μM). The cellular uptake of DOX increased upon addition of free CQ, indicating that CQ may interact with P-gp and MRP1; however, the expressions of P-gp and MRP1 remained unchanged. In contrast, the expression of the autophagy-related protein LC3-II increased remarkably. Therefore, the mechanism of MDR reversal may be closely related to autophagic inhibition. Evaluation of anti-tumor activity was achieved in an MCF-7/ADR multicellular tumor spheroid model and transgenic zebrafish model. DOX/CQ co-encapsulated liposome exerted a better anti-tumor effect in both models than that of liposomal DOX or DOX alone. These findings suggest that encapsulating CQ with DOX in liposomes significantly improves the sensitivity of DOX in DOX-resistant cancer cells.     

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of mdrl ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDRl ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzedin vitro andin vivo; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDRl ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effective treatment method for patients with CEA-producing lung cancers which was usually refractory to conventional chemotherapy     

Opposing effects of 15-lipoxygenase-1 and -2 metabolites on MAPK signaling in prostate. Alteration in peroxisome proliferator-activated receptor gamma

Human prostate tumors have elevated levels of 15-lipoxygenase-1 (15-LOX-1) and data suggest that 15-LOX-1 may play a role in the development of prostate cancer. In contrast, 15-LOX-2 expression is higher in normal rather than in tumor prostate tissue and appears to suppress cancer development. We recently reported that 13-(S)-HODE, the 15-LOX-1 metabolite, up-regulates the MAP kinase signaling pathway and subsequently down-regulates PPARgamma in human colorectal carcinoma cells. To determine whether this mechanism is applicable to prostate cancer and what the effects of 15-LOX-2 are, we investigated the effect of 15-LOX-1, 15-LOX-2, and their metabolites on epidermal growth factor (EGF)- and insulin-like growth factor (IGF)-1 signaling in prostate carcinoma cells. In PC3 cells, 13-(S)-HODE, a 15-LOX-1 metabolite, up-regulated MAP kinase while in contrast 15-(S)-HETE, a 15-LOX-2 metabolite, down-regulated MAP kinase. As a result, 13-(S)-HODE increased PPARgamma phosphorylation while a subsequent decrease in PPARgamma phosphorylation was observed with 15-(S)-HETE. Thus, 15-LOX metabolites have opposing effects on the regulation of the MAP kinase signaling pathway and a downstream target of MAP kinase signaling like PPARgamma. In addition to the EGF signaling pathway, the IGF signaling pathway appears to be linked to prostate cancer. 13-(S)-HODE and 15-(S)-HETE up-regulate or down-regulate, respectively, both the MAPK and Akt pathways after activation with IGF-1. Thus, the effect of these lipid metabolites is not solely restricted to EGF signaling and not solely restricted to MAPK signaling. These results provide a plausible mechanism to explain the apparent opposing effects 15-LOX-1 and 15-LOX-2 play in prostate cancer.     

Identification of a novel mechanism for reversal of doxorubicin-induced chemotherapy resistance by TXNIP in triple-negative breast cancer via promoting reactive oxygen-mediated DNA damage

Given that triple-negative breast cancer (TNBC) lacks specific receptors (estrogen and progesterone receptors and human epidermal growth factor receptor 2) and cannot be treated with endocrine therapy, chemotherapy has remained the mainstay of treatment. Drug resistance is reportedly the main obstacle to the clinical use of doxorubicin (DOX) in this patient population. Accordingly, screening molecules related to chemoresistance and studying their specific mechanisms has clinical significance for improving the efficacy of chemotherapy in TNBC patients. Thioredoxin-interacting protein (TXNIP) is a metabolism-related protein that plays a tumor suppressor role in various malignant tumors; however, the specific role of TXNIP in tumor chemoresistance has not been reported. In the present study, we explored the potential molecular mechanism of TXNIP in the chemoresistance of TNBC for the first time. The results showed that TXNIP inhibited the proliferation of TNBC drug-resistant cells and promoted apoptosis in vitro and in vivo. Furthermore, TXNIP promoted the synthesis of reactive oxygen species (ROS) and the accumulation of DNA damage caused by DOX and increased γ-H2AX levels in a time and dose-dependent manner. Moreover, ROS scavenger pretreatment could block DNA damage induced by TXNIP and restore the resistance of TNBC resistant cells to DOX to a certain extent. In addition, we found that the small molecule c-Myc inhibitor 10058-F4 promoted TXNIP expression, increased ROS synthesis in cells, and could enhance the cytotoxicity of chemotherapy drugs in vitro and in vivo when combined with DOX. These results indicated that c-Myc inhibitor 10058-F4 could induce TXNIP upregulation in TNBC drug-resistant cells, and the upregulated TXNIP increased the accumulation of ROS-dependent DNA damage, thereby decreasing chemotherapy resistance of TNBC. Our findings reveal a new mechanism of mediating drug resistance and provide a new drug combination strategy to overcome DOX resistance in TNBC.Subject terms: Breast cancer, Cancer therapeutic resistance     

A gene cluster encoding human epidermis-type lipoxygenases at chromosome 17p13.1: cloning, physical mapping, and expression

Epidermis-type lipoxygenases, a distinct subclass within the multigene family of mammalian lipoxygenases (LOX), comprise recently discovered novel isoenzymes isolated from human and mouse skin including human 15-LOX-2, human and mouse 12R-LOX, mouse 8S-LOX, and mouse e-LOX-3. We have isolated the human homologue of mouse e-LOX-3. The cDNA of 3362 bp encodes a 711-amino-acid protein displaying 89% sequence identity with the mouse protein and exhibiting the same unusual structural feature, i.e., an extra segment of 41 amino acids, which can be located beyond the N-terminal beta-barrel domain at the surface of the C-terminal catalytic domain. The gene encoding e-LOX-3, ALOXE3, was found to be part of a gene cluster of approximately 100 kb on human chromosome 17p13.1 containing in addition the 12R-LOX gene, ALOX12B, the 15-LOX-2 gene, ALOX15B, and a novel 15-LOX pseudogene, ALOX15P. ALOXE3 and ALOX12B are arranged in a head-to-tail fashion separated by 8.5 kb. The genes are split into 15 exons and 14 introns spanning 22 and 15 kb, respectively. ALOX15P was found on the opposite DNA strand directly adjacent to the 3'-untranslated region of ALOX12B. ALOX15B is located in the same orientation 25 kb downstream of ALOX12B, and is composed of 14 exons and 13 introns spanning a total of 9.7 kb of genomic sequence. RT-PCR analysis demonstrated a predominant expression of ALOXE3, ALOX12B, and ALOX15B in skin.     

Overexpression of 15-lipoxygenase-1 induces growth arrest through phosphorylation of p53 in human colorectal cancer cells

To investigate the function of 15-lipoxygenase-1 (15-LOX-1) in human colorectal cancer, we overexpressed 15-LOX-1 in HCT-116 human colorectal cancer cells. Clones expressing the highest levels of 15-LOX-1 displayed reduced viability compared with the HCT-116-Vector control cells. Further, by cell cycle gene array analyses, the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and MDM2 genes were up-regulated in 15-LOX-1-overexpressing cells. The induction of p21(WAF1/CIP1) and MDM2 were linked to activation of p53 by 15-LOX-1, as there was a dramatic induction of phosphorylated p53 (Ser15) in 15-LOX-1-overesxpressing cells. However, the 15-LOX-1 metabolites 13(S)-hydroxyoctadecadienoic acid and 15(S)-hydroxyeicosatetraenoic acid failed to induce phosphorylation of p53 at Ser15, and the 15-LOX-1 inhibitor PD146176 did not inhibit the phosphorylation of p53 at Ser15 in 15-LOX-1-overexpressing cells. Nonetheless, the growth-inhibitory effects of 15-LOX-1 were p53 dependent, as 15-LOX-1 overexpression had no effect on cell growth in p53 (-/-) HCT-116 cells. Finally, treatment of HCT-116-15-LOX-1 cells with different kinase inhibitors suggested that the effects of 15-LOX-1 on p53 phosphorylation and activation were due to effects on DNA-dependent protein kinase. Collectively, these findings suggest a new mechanism to explain the biological activity of 15-LOX-1, where 15-LOX plays a stoichiometric role in activating a DNA-dependent protein kinase-dependent pathway that leads to p53-dependent growth arrest.     

Reversal of cisplatin and multidrug resistance by ribozyme-mediated glutathione suppression

gamma-Glutamylcysteine synthetase (gamma-GCS) is a key enzyme in glutathione (GSH) synthesis, and is thought to play a significant role in intracellular detoxification, especially of anticancer drugs. Increased levels of GSH are commonly found in the drug-resistant human cancer cells. We designed a hammerhead ribozyme against gamma-GCS mRNA (anti-gamma-GCS Rz), which specifically down-regulated gamma-GCS gene expression in the HCT-8 human colon cancer cell line. The aim of this study was to reverse the cisplatin and multidrug resistance for anticancer drugs. The cisplatin-resistant HCT-8 cells (HCT-8DDP cells) overexpressed MRP and MDR1 genes, and showed resistance to not only cisplatin (CDDP), but also doxorubicin (DOX) and etoposide (VP-16). We transfected a vector expressing anti-gamma-GCS Rz into the HCT-8DDP cells (HCT-8DDP/Rz). The anti-gamma-GCS Rz significantly suppressed MRP and MDR, and altered anticancer drug resistance. The HCT-8DDP/Rz cells were more sensitive to CDDP, DOX and VP-16 by 1.8-, 4.9-, and 1.5-fold, respectively, compared to HCT-8DDP cells. The anti-gamma-GCS Rz significantly down-regulated gamma-GCS gene expression as well as MRP/MDR1 expression, and reversed resistance to CDDP, DOX and VP-16. These results suggested that gamma-GCS plays an important role in both cisplatin and multidrug resistance in human cancer cells.     

15-LOX-1 inhibits p21 (Cip/WAF 1) expression by enhancing MEK-ERK 1/2 signaling in colon carcinoma cells

Currently, some controversy exists regarding the precise role of 15-lipoxygenase-1 (15-LOX-1) in colorectal carcinogenesis and other aspects of cancer biology. The aim of this study was to evaluate the effect of 15-LOX-1 on p21 (Cip/WAF 1) expression and growth regulation in human colon carcinoma cells. The effect of 13-S-hydroxyoctadecadienoic acid (HODE), a product of 15-LOX-1, on p21 (Cip/WAF 1) expression was evaluated in Caco-2 cells treated with sodium butyrate (NaBT) and/or nordihydroguaiarectic acid (NDGA), a LOX inhibitor. The effect of transfecting HCT-116 cells with 15-LOX-1 was also examined. NaBT-induced p21 (Cip/WAF 1) expression was enhanced by treatment with NDGA and 13-S-HODE reversed NaBT-induced p21 (Cip/WAF 1) expression in Caco-2 cells. Overexpression of 15-LOX-1 induced extracellular signal-related kinase (ERK) 1/2 phosphorylation, decreased p21 (Cip/WAF 1) expression, and increased HCT-116 cell growth. Treatment with NDGA decreased ERK 1/2 phosphorylation, and increased p21 (Cip/WAF 1) expression in 15-LOX-1 overexpressing HCT-116 cells. Our experimental results support the hypothesis that 15-LOX-1 may have "pro-neoplastic" effects during the development of colorectal cancer.     

Drug-resistant cancer cell-derived exosomal EphA2 promotes breast cancer metastasis via the EphA2-Ephrin A1 reverse signaling

Tumor metastasis induced by drug resistance is a major challenge in successful cancer treatment. Nevertheless, the mechanisms underlying the pro-invasive and metastatic ability of drug resistance remain elusive. Exosome-mediated intercellular communications between cancer cells and stromal cells in tumor microenvironment are required for cancer initiation and progression. Recent reports have shown that communications between cancer cells also promote tumor aggression. However, little attention has been regarded on this aspect. Herein, we demonstrated that drug-resistant cell-derived exosomes promoted the invasion of sensitive breast cancer cells. Quantitative proteomic analysis showed that EphA2 was rich in exosomes from drug-resistant cells. Exosomal EphA2 conferred the invasive/metastatic phenotype transfer from drug-resistant cells to sensitive cells. Moreover, exosomal EphA2 activated ERK1/2 signaling through the ligand Ephrin A1-dependent reverse pathway rather than the forward pathway, thereby promoting breast cancer progression. Our findings indicate the key functional role of exosomal EphA2 in the transmission of aggressive phenotype between cancer cells that do not rely on direct cell–cell contact. Our study also suggests that the increase of EphA2 in drug-resistant cell-derived exosomes may be an important mechanism of chemotherapy/drug resistance-induced breast cancer progression.Subject terms: Breast cancer, Metastasis     

Formulation and Optimization of Doxorubicin and Biochanin A Combinational Liposomes for Reversal of Chemoresistance

Circumvention of drug resistance still remains a challenge in the development of anticancer therapeutics. Combinational nano-formulations provide many avenues for effective cancer therapy and reversal of drug resistance. In the current study, combination of biochanin A (BioA) and doxorubicin (DOX) in liposomes were prepared and studied for its potential to reverse DOX resistance in COLO205 cells. After development and validation of DOX resistant cells of COLO205 (ColoR), dosing ratio of DOX and BioA for reversal of DOX resistance was determined by co-treatment in ColoR cells. As limited solubility and analytical data available for BioA, therefore solubility was studied for BioA and analytical method was developed for the combination. Combinational liposomes were prepared and optimized for both lipid content and surface charge by evaluating size, polydispersity index, zeta potential, and encapsulation efficiency. The optimized formulation had a size about 125 nm; zeta potential of ?19.5 mV and 70% encapsulation efficiency (EE) for BioA. Thus, prepared combinational liposomes of DOX and BioA were evaluated for its cellular uptake and efficacy to reverse DOX resistance. From the study, increased DOX uptake and promising effect for reversal of DOX resistance was observed.